Cytosolic phospholipase A2-beta enzymes

ABSTRACT

The invention provides a novel calcium-independent cytosolic phospholipase A 2 -Beta enzyme, polynucleotides encoding such enzyme and methods for screening unknown compounds for anti-inflammatory activity mediated by the arachidonic acid cascade.

The present invention relates to a purified cytosolic phospholipaseA₂-Beta (cPLA₂-β) enzymes which are useful for assaying chemical agentsfor anti-inflammatory activity.

BACKGROUND OF THE INVENTION

The phospholipase A₂ enzymes comprise a widely distributed family ofenzymes which catalyze the hydrolysis of the acyl ester bond ofglycerophospholipids at the sn-2 position. One kind of phospholipase A₂enzymes, secreted phospholipase A₂ or sPLA₂, are involved in a number ofbiological functions, including phospholipid digestion, the toxicactivities of numerous venoms, and potential antibacterial activities. Asecond kind of phospholipase A₂ enzymes, the intracellular phospholipaseA₂ enzymes, also known as cytosolic phospholipase A₂ or cPLA₂, areactive in membrane phospholipid turnover and in regulation ofintracellular signalling mediated by the multiple components of thewell-known arachidonic acid cascade. One or more cPLA₂ enzymes arebelieved to be responsible for the rate limiting step in the arachidonicacid cascade, namely, release of arachidonic acid from membraneglycerophospholipids. The action of cPLA₂ also results in biosynthesisof platelet activating factor (PAF). U.S. Pat. Nos. 5,322,776,5,354,677, 5,527,698 and 5,593,878 disclose such enzymes (sometimesreferred to herein as “cPLA₂α”).

The phospholipase B enzymes are a family of enzymes which catalyze thehydrolysis of the acyl ester bond of glycerophospholipids at the sn-1and sn-2 positions. The mechanism of hydrolysis is unclear but mayconsist of initial hydrolysis of the sn-2 fatty acid followed by rapidcleavage of the sn-1 substituent, i.e., functionally equivalent to thecombination of phospholipase A₂ and lysophospholipase (Saito et al.,Methods of Enzymol., 1991, 197, 446; Gassama-Diagne et al., J. Biol.Chem., 1989, 264, 9470). Whether these two events occur at the same ortwo distinct active sites has not been resolved. It is also unknown ifthese enzymes have a preference for the removal of unsaturated fattyacids, in particular arachidonic acid, at the sn-2 position andaccordingly contribute to the arachidonic acid cascade.

Upon release from the membrane, arachidonic acid may be metabolized viathe cyclooxygenase pathway to produce the various prostaglandins andthromboxanes, or via the lipoxygenase pathway to produce the variousleukotrienes and related compounds. The prostaglandins, leukotrienes andplatelet activating factor are well known mediators of variousinflammatory states, and numerous anti-inflammatory drugs have beendeveloped which function by inhibiting one or more steps in thearachidonic acid cascade. The efficacy of the present anti-inflammatorydrugs which act through inhibition of arachidonic acid cascade steps islimited by the existence of side effects which may be harmful to variousindividuals.

A very large industrial effort has been made to identify additionalanti-inflammatory drugs which inhibit the arachidonic acid cascade. Ingeneral, this industrial effort has employed the secreted phospholipaseA₂ enzymes in inhibitor screening assays, for example, as disclosed inU.S. Pat. No. 4,917,826. However, because the secreted phospholipase A₂enzymes are extracellular proteins (i.e., not cytosolic) and do notselectively hydrolyze arachidonic acid, they are presently not believedto contribute to prostaglandin and leukotriene production. While someinhibitors of the small secreted phospholipase A₂ enzymes have beenreported to display anti-inflammatory activity, such as bromphenacylbromide, mepacrine, and certain butyrophenones as disclosed in U.S. Pat.No. 4,239,780. The site of action of these compounds is unclear as theseagents retain anti-inflammatory activity in mouse strains lacking sPLA₂.It is presently believed that inhibitor screening assays should employcytosolic phospholipase A₂ enzymes which initiate the arachidonic acidcascade.

An improvement in the search for anti-inflammatory drugs which inhibitthe arachidonic acid cascade was developed in commonly assigned U.S.Pat. No. 5,322,776, incorporated herein by reference. In thatapplication, a cytosolic form of phospholipase A₂ was identified,isolated, and cloned. Use of the cytosolic form of phospholipase. A₂ toscreen for anti-inflammatory drugs provides a significant improvement inidentifying inhibitors of the arachidonic acid cascade. The cytosolicphospholipase A₂ disclosed in U.S. Pat. No. 5,322,776 is a 110 kDprotein which depends on the presence of elevated levels of calciuminside the cell for its activity. The cPLA₂ of U.S. Pat. No. 5,322,776plays a pivotal role in the production of leukotrienes andprostaglandins initiated by the action of pro-inflammatory cytokines andcalcium mobilizing agents. The cPLA₂ of U.S. Pat. No. 5,322,776 isactivated by phosphorylation on serine residues and increasing levels ofintracellular calcium, resulting in translocation of the enzyme from thecytosol to the membrane where arachidonic acid is selectively hydrolyzedfrom membrane phospholipids.

In addition to the cPLA₂ of U.S. Pat. No. 5,322,776, some cells containcalcium independent phospholipase A₂/B enzymes. For example, suchenzymes have been identified in rat, rabbit, canine and human hearttissue (Gross, T C M, 1991, 2, 115; Zupan et al., J. Med. Chem., 1993,36, 95; Hazen et al., J. Clin. Invest., 1993, 91, 2513; Lehman et al.,J. Biol. Chem., 1993, 268, 20713; Zupan et al., J. Biol. Chem., 1992,267, 8707; Hazen et al., J. Biol. Chem., 1991, 266, 14526; Loeb et al.,J. Biol. Chem., 1986, 261, 10467; Wolf et al., J. Biol. Chem., 1985,260, 7295; Hazen et al., Meth. Enzymol., 1991, 197, 400; Hazen et al.,J. Biol. Chem., 1990, 265, 10622; Hazen et al., J. Biol. Chem., 1993,268, 9892; Ford et al., J. Clin. Invest., 1991, 88, 331; Hazen et al.,J. Biol. Chem., 1991, 266, 5629; Hazen et al., Circulation Res., 1992,70, 486; Hazen et al., J. Biol. Chem., 1991, 266, 7227; Zupan et al.,FEBS, 1991, 284, 27), as well as rat and human pancreatic islet cells(Ramanadham et al., Biochemistry, 1993, 32, 337; Gross et al.,Biochemistry, 1993, 32, 327), in the macrophage-like cell line, P388D₁(Ulevitch et al., J. Biol. Chem., 1988, 263, 3079; Ackermann et al., J.Biol. Chem., 1994, 269, 9227; Ross et al., Arch. Biochem. Biophys.,1985, 238, 247; Ackermann et al., FASEB Journal, 1993, 7(7), 1237), invarious rat tissue cytosols (Nijssen et al., Biochim. Biophys. Acta,1986, 876, 611; Pierik et al., Biochim. Biophys. Acta, 1988, 962, 345;Aarsman et al., J. Biol. Chem., 1989, 264, 10008), bovine brain (Ueda etal., Biochem. Biophys, Res. Comm., 1993, 195, 1272; Hirashima et al., J.Neurochem., 1992, 59, 708), in yeast (Saccharomyces cerevisiae)mitochondria (Yost et al., Biochem. International, 1991, 24, 199),hamster heart cytosol (Cao et al., J. Biol. Chem., 1987, 262, 16027),rabbit lung microsomes (Angle et al., Biochim. Biophys. Acta, 1988, 962,234) and guinea pig intestinal brush-border membrane (Gassama-Diagne etal., J. Biol. Chem., 1989, 264, 9470). U.S. Pat. Nos. 5,466,595,5,554,511 and 5,589,170 also disclose calcium independent cPLA₂/Benzymes (sometimes referred to herein as “iPLA₂”).

It is believed that the phospholipase enzymes may perform importantfunctions in release of arachidonic acid in specific tissues which arecharacterized by unique membrane phospholipids, by generatinglysophospholipid species which are deleterious to membrane integrity orby remodeling of unsaturated species of membrane phospholipids throughdeacylation/reacylation mechanisms. The activity of such a phospholipasemay well be regulated by mechanisms that are different from that of thecPLA₂ of U.S. Pat. No. 5,322,776. In addition the activity may be morepredominant in certain inflamed tissues over others.

Therefore, it would, be desirable to identify and isolate additionalcPLA₂ enzymes.

SUMMARY OF THE INVENTION

In other embodiments, the invention provides isolated polynucleotidescomprising a nucleotide sequence selected from the group consisting of:

-   -   (a) the nucleotide sequence of SEQ ID NO:1;    -   (b) a nucleotide sequence encoding the amino acid sequence of        SEQ ID NO:2;    -   (c) a nucleotide sequence encoding a fragment of the amino acid        sequence of SEQ ID NO:2 having activity in a mixed micelle assay        with 1-palmitoyl-2-[¹⁴C]-arachidonyl-phosphatidylcholine;    -   (d) a nucleotide sequence capable of hybridizing with the        sequence of (a), (b) or (c) which encodes a peptide having        activity in a mixed micelle assay with        1-palmitoyl-2-[¹⁴C]-arachidonyl-phosphatidylcholine;    -   (e) allelic variants of the sequence of (a);    -   (f) the nucleotide sequence of SEQ ID NO:3;    -   (g) a nucleotide sequence encoding the amino acid sequence of        SEQ ID NO:4;    -   (h) a nucleotide sequence encoding a fragment of the amino acid        sequence of SEQ ID NO:4 having activity in a mixed micelle assay        with 1-palmitoyl-2-[¹⁴C]-arachidonyl-phosphatidylcholine; and    -   (i) a nucleotide sequence capable of hybridizing with the        sequence of (f), (g) or (h) which encodes a peptide having        activity in a mixed micelle assay with        1-palmitoyl-2[¹⁴C]-arachidonyl-phosphatidylcholine.

Expression vectors comprising such polynucleotides and host cellstransformed with such vectors are also provided by the presentinvention. Compositions comprising peptides encoded by suchpolynucleotides are also provided.

The present invention also provides processes for producing aphospholipase enzyme, said process comprising: (a) establishing aculture of the host cell transformed with a cPLA₂-Beta encodingpolynucleotide in a suitable culture medium; and (b) isolating saidenzyme from said culture. Compositions comprising a peptide madeaccording to such processes are also provided.

Certain embodiments of the present invention provide compositionscomprising a peptide comprising an amino acid sequence selected from thegroup consisting of:

-   -   (a) the amino acid sequence of SEQ ID NO:2;    -   (b) a fragment of the amino acid sequence of SEQ ID NO:2 having        activity in a mixed micelle assay with        1-palmitoyl-2-[¹⁴C]-arachidonyl-phosphatidylcholine;    -   (c) the amino acid sequence of SEQ ID NO:4; and    -   (d) a fragment of the amino acid sequence of SEQ ID NO:4 having        activity in a mixed micelle assay with        1-palmitoyl-2-[¹⁴C]-arachidonyl-phosphatidylcholine.

The present invention also provides methods for identifying an inhibitorof phospholipase activity said method comprising: (a) combining aphospholipid, a candidate inhibitor compound, and a compositioncomprising a phospholipase enzyme peptide; and (b) observing whethersaid phospholipase enzyme peptide cleaves said phospholipid and releasesfatty acid thereby, wherein the peptide composition is one of thosedescribed above. Inhibitor of phospholipase activity identified by suchmethods, pharmaceutical compositions comprising a therapeuticallyeffective amount of such inhibitors and a pharmaceutically acceptablecarrier, and methods of reducing inflammation by administering suchpharmaceutical compositions to a mammalian subject are also provided.

Polyclonal and monoclonal antibodies to the peptides of the inventionare also provided.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A presents data evidenceing increased PLA₂ activity in cellstransfected with pEDΔC-n48.

FIG. 1B presents data comparing PLA₂ activities of cells transfectedwith plasmids expressing, cPLA₂α, cPLA₂β and iPLA₂.

FIG. 2 depicts a gel evidencing the expression of cPLA₂β in COS cells.

DETAILED DESCRIPTION OF THE INVENTION

A cDNA encoding the cPLA₂-Beta of the present invention was isolated asdescribed in Example 1. The sequence of the partial cDNA first isolatedis reported as SEQ ID NO:1. The amino acid sequence encoded by such cDNAis SEQ ID NO:2. For purposes of expression, as explained in Example 1,polynucleotides encoding N-terminal sequence from cPLA₂ was added to thepartial cDNA. The polynucleotide sequence of this fusion is reported asSEQ ID NO:3. The amino acid sequence encoded by the fuion cDNA isreported as SEQ ID NO:4.

The invention also encompasses allelic variations of the cDNA sequenceas set forth in SEQ ID NO:1 and SEQ ID NO:3, that is,naturally-occurring alternative forms of the cDNAs of SEQ ID NO:1 andSEQ ID NO:3 which also encode phospholipase enzymes of the presentinvention. Also included in the invention are isolated DNAs whichhybridize to the DNA sequence set forth in SEQ ID NO:1 or SEQ ID NO:3under stringent (e.g. 4×SSC at 65° C. or 50% formamide and 4×SSC at 42°C.), or relaxed (4×SSC at 50° C. or 30-40% formamide at 42° C.)conditions.

The isolated polynucleotides of the invention may be operably linked toan expression control sequence such as the pMT2 or pED expressionvectors disclosed in Kaufman et al., Nucleic Acids Res. 19, 4485-4490(1991), in order to produce the phospholipase enzyme peptidesrecombinantly. Many suitable expression control sequences are known inthe art. General methods of expressing recombinant proteins are alsoknown and are exemplified in R. Kaufman, Methods in Enzymology 185,537-566 (1990). As defined herein “operably linked” means enzymaticallyor chemically ligated to form a covalent bond between the isolatedpolynucleotide of the invention and the expression control sequence, insuch a way that the phospholipase enzyme peptide is expressed by a hostcell which has been transformed (transfected) with the ligatedpolynucleotide/expression control sequence.

A number of types of cells may act as suitable host cells for expressionof the phospholipase enzyme peptide. Suitable host cells are capable ofattaching carbohydrate side chains characteristic of functionalphospholipase enzyme peptide. Such capability may arise by virtue of thepresence of a suitable glycosylating enzyme within the host cell,whether naturally occurring, induced by chemical mutagenesis, or throughtransfection of the host cell with a suitable expression plasmidcontaining a polynucleotide encoding the glycosylating enzyme. Hostcells include, for example, monkey COS cells, Chinese Hamster Ovary(CHO) cells, human kidney 293 cells, human epidermal A431 cells, humanColo205 cells, 3T3 cells, CV-1 cells, other transformed primate celllines, normal diploid cells, cell strains derived from in vitro cultureof primary tissue, primary explants, HeLa cells, mouse L cells, BHK,HL-60, U937, or HaK cells.

The phospholipase enzyme peptide may also be produced by operablylinking the isolated polynucleotide of the invention to suitable controlsequences in one or more insect expression vectors, and employing aninsect expression system. Materials and methods for baculovirus/insectcell expression systems are commercially available in kit form from,e.g., Invitrogen, San Diego, Calif., U.S.A. (the MaxBac® kit), and suchmethods are well known in the art, as described in Summers and Smith,Texas Agricultural Experiment Station Bulletin No. 1555 (1987),incorporated herein by reference.

Alternatively, it may be possible to produce the phospholipase enzymepeptide in lower eukaryotes such as yeast or in prokaryotes such asbacteria. Potentially suitable yeast strains include Saccharomycescerevisiae, Schizosaccharomyces pombe, Kluyveromyces strains, Candida,or any yeast strain capable of expressing heterologous proteins.Potentially suitable bacterial strains include Escherichia coli,Bacillus subtilis, Salmonella typhimurium, or any bacterial straincapable of expressing heterologous proteins. If the phospholipase enzymepeptide is made in yeast or bacteria, it is necessary to attach theappropriate carbohydrates to the appropriate sites on the protein moietycovalently, in order to obtain the glycosylated phospholipase enzymepeptide. Such covalent attachments may be accomplished using knownchemical or enzymatic methods.

The phospholipase enzyme peptide of the invention may also be expressedas a product of transgenic animals, e.g., as a component of the milk oftransgenic cows, goats, pigs, or sheep which are characterized bysomatic or germ cells containing a polynucleotide encoding thephospholipase enzyme peptide.

The phospholipase enzyme peptide of the invention may be prepared byculturing transformed host cells under culture conditions necessary toexpress a phospholipase enzyme peptide of the present invention. Theresulting expressed protein may then be purified from culture medium orcell extracts as described in the examples below.

Alternatively, the phospholipase enzyme peptide of the invention isconcentrated using a commercially available protein concentrationfilter, for example, an Amicon or Millipore Pellicon ultrafiltrationunit. Following the concentration step, the concentrate can be appliedto a purification matrix such as a gel filtration medium. Alternatively,an anion exchange resin can be employed, for example, a matrix orsubstrate having pendant diethylaminoethyl (DEAE) groups. The matricescan be acrylamide, agarose, dextran, cellulose or other types commonlyemployed in protein purification. Alternatively, a cation exchange stepcan be employed. Suitable cation exchangers include various insolublematrices comprising sulfopropyl or carboxymethyl groups. Sulfopropylgroups are preferred (e.g., S-Sepharose® columns). The purification ofthe phospholipase enzyme peptide from culture supernatant may alsoinclude one or more column steps over such affinity resins asconcanavalin A-agarose, heparin-toyopearl® or Cibacrom blue 3GASepharose®; or by hydrophobic interaction chromatography using suchresins as phenyl ether, butyl ether, or propyl ether; or byimmunoaffinity chromatography.

Finally, one or more reverse-phase high performance liquidchromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media,e.g., silica gel having pendant methyl or other aliphatic groups, can beemployed to further purify the phospholipase enzyme peptide. Some or allof the foregoing purification steps, in various combinations, can alsobe employed to provide a substantially homogeneous isolated recombinantprotein. The phospholipase enzyme peptide thus purified is substantiallyfree of other mammalian proteins and is defined in accordance with thepresent invention as “isolated phospholipase enzyme peptide”.

The cPLA₂-Beta of the present invention may be used to screen forcompounds having anti-inflammatory activity mediated by the variouscomponents of the arachidonic acid cascade. Many assays forphospholipase activity are known and may be used with the phospholipaseA₂-Beta on the present invention to screen unknown compounds. Forexample, such an assay may be a mixed micelle assay as described inExample 2. Other known phospholipase activity assays include, withoutlimitation, those disclosed in U.S. Pat. No. 5,322,776. These assays maybe performed manually or may be automated or robotized for fasterscreening. Methods of automation and robotization are known to thoseskilled in the art.

In one possible screening assay, a first mixture is formed by combininga phospholipase enzyme peptide of the present invention with aphospholipid cleavable by such peptide, and the amount of hydrolysis inthe first mixture (B₀) is measured. A second mixture is also formed bycombining the peptide, the phospholipid and the compound or agent to bescreened, and the amount of hydrolysis in the second mixture (B) ismeasured. The amounts of hydrolysis in the first and second mixtures arecompared, for example, by performing a B/B_(o) calculation. A compoundor agent is considered to be capable of inhibiting phospholipaseactivity (i.e., providing anti-inflammatory activity) if a decrease inhydrolysis in the second mixture as compared to the first mixture isobserved. The formulation and optimization of mixtures is within thelevel of skill in the art, such mixtures may also contain buffers andsalts necessary to enhance or to optimize the assay, and additionalcontrol assays may be included in the screening assay of the invention.

Other uses for the cPLA₂-Beta of the present invention are in thedevelopment of monoclonal and polyclonal antibodies. Such antibodies maybe generated by employing purified forms of the cPLA₂ or immunogenicfragments thereof as an antigen using standard methods for thedevelopment of polyclonal and monoclonal antibodies as are known tothose skilled in the art. Such polyclonal or monoclonal antibodies areuseful as research or diagnostic tools, and further may be used to studyphospholipase A₂ activity and inflammatory conditions.

Pharmaceutical compositions containing anti-inflammatory agents (i.e.,inhibitors) identified by the screening method of the present inventionmay be employed to treat, for example, a number of inflammatoryconditions such as rheumatoid arthritis, psoriasis, asthma, inflammatorybowel disease and other diseases mediated by increased levels ofprostaglandins, leukotriene, or platelet activating factor.Pharmaceutical compositions of the invention comprise a therapeuticallyeffective amount of a cPLA₂ inhibitor compound first identifiedaccording to the present invention in a mixture with an optionalpharmaceutically acceptable carrier. The term “pharmnaceuticallyacceptable” means a non-toxic material that does not interfere with theeffectiveness of the biological activity of the active ingredient(s).The term “therapeutically effective amount” means the total amount ofeach active component of the method or composition that is sufficient toshow a meaningful patient benefit, i.e., healing or amelioration ofchronic conditions or increase in rate of healing or amelioration. Whenapplied to an individual active ingredient, administered alone, the termrefers to that ingredient alone. When applied to a combination, the termrefers to combined amounts of the active ingredients that result in thetherapeutic effect, whether administered in combination, serially orsimultaneously. A therapeutically effective dose of the inhibitor ofthis invention is contemplated to be in the range of about 0.1 μg toabout 100 mg per kg body weight per application. It is contemplated thatthe duration of each application of the inhibitor will be in the rangeof 12 to 24 hours of continuous administration. The characteristics ofthe carrier or other material will depend on the route ofadministration.

The amount of inhibitor in the pharmaceutical composition of the presentinvention will depend upon the nature and severity of the conditionbeing treated, and on the nature of prior treatments which the patienthas undergone. Ultimately, the attending physician will decide theamount of inhibitor with which to treat each individual patient.Initially, the attending physician will administer low doses ofinhibitor and observe the patient's response. Larger doses of inhibitormay be administered until the optimal therapeutic effect is obtained forthe patient, and at that point the dosage is not increased further.

Administration is preferably intravenous, but other known methods ofadministration for anti-inflammatory agents may be used. Administrationof the anti-inflammatory compounds identified by the method of theinvention can be carried out in a variety of conventional ways. Forexample, for topical administration, the anti-inflammatory compound ofthe invention will be in the form of a pyrogen-free, dermatologicallyacceptable liquid or semi-solid formulation such as an ointment, cream,lotion, foam or gel. The preparation of such topically appliedformulations is within the skill in the art. Gel formulation shouldcontain, in addition to the anti-inflammatory compound, about 2 to about5% W/W of a gelling agent. The gelling agent may also function tostabilize the active ingredient and preferably should be water soluble.The formulation should also contain about 2% W/V of a bactericidal agentand a buffering agent. Exemplary gels include ethyl, methyl, and propylcelluloses. Preferred gels include carboxypolymethylene such as Carbopol(934P; B.F. Goodrich), hydroxypropyl methylcellulose phthalates such asMethocel (K100M premium; Merril Dow), cellulose gums such as Blanose(7HF; Aqualon, U.K.), xanthan gums such as Keltrol (TF; KelkoInternational), hydroxyethyl cellulose oxides such as Polyox (WSR 303;Union Carbide), propylene glycols, polyethylene glycols and mixturesthereof. If Carbopol is used, a neutralizing agent, such as NaOH, isalso required in order to maintain pH in the desired range of about 7 toabout 8 and most desirably at about 7.5. Exemplary preferredbactericidal agents include steryl alcohols, especially benzyl alcohol.The buffering agent can be any of those already known in the art asuseful in preparing medicinal formulations, for example 20 mM phosphatebuffer, pH 7.5.

Cutaneous or subcutaneous injection may also be employed and in thatcase the anti-inflammatory compound of the invention will be in the formof pyrogen-free, parenterally acceptable aqueous solutions. Thepreparation of such parenterally acceptable solutions, having due regardto pH, isotonicity, stability, and the like, is within the skill in theart.

Intravenous injection may be employed, wherein the anti-inflammatorycompound of the invention will be in the form of pyrogen-free,parenterally acceptable aqueous solutions. A preferred pharmaceuticalcomposition for intravenous injection should contain, in addition to theanti-inflammatory compound, an isotonic vehicle such as Sodium ChlorideInjection, Ringer's Injection, Dextrose Injection, Dextrose and SodiumChloride Injection, Lactated Ringer's Injection, or other vehicle asknown in the art. The pharmaceutical composition according to thepresent invention may also contain stabilizers, preservatives, buffers,antioxidants, or other additive known to those of skill in the art.

The amount of anti-inflammatory compound in the pharmaceuticalcomposition of the present invention will depend upon the nature andseverity of the condition being treated, and on the nature of priortreatments which the patient has undergone. Ultimately, the attendingphysician will decide the amount of anti-inflammatory compound withwhich to treat each individual patient.

Anti-inflammatory compounds identified using the method of the presentinvention may be administered alone or in combination with otheranti-inflammation agents and therapies.

EXAMPLE 1

Library Construction

Oligo-dT primed and random primed cDNA libraries were constructed fromU937 cells using a Poly A Track kit for isolation of mRNA (Promega), aSuperscript Choice kit for the generation of double stranded cDNA (GibcoBRL), and a Lambda ZapII phage cloning kit (Stratagene).

Clone Identification

Two cPLA₂-β specific deoxyribonucleotides were designed based on thesequence of EST clone W92213: 5′-CCTCCTGCAGCCCACTCGGGAC-3′ (SEQ ID NO:5)5′-GCTGACCAGAGGAAAGTGCAGC-3′ (SEQ ID NQ:6)These oligonucleotides were used to screen 10⁶ recombinates of both theoligo dT primed and random primed library. One clone which hybridizeswith both oligonucleotides, clone 52A, was examined for complete DNAsequence determination (SEQ ID NO:1). The partial coding sequence onthis clone begins at nucleotide 1560 and continues to a stop codon atnucleotide 3894, representing 778 amino acids (see SEQ ID NO:2). Theregion on the DNA sequence 5′ to nucleotide 1560 fits a splice acceptorconsensus sequence and is therefore assumed to be unspliced intronsequence.

A comparison of the previous cPLA₂ amino acid sequence with thepredicted cPLA₂-β sequence reveals 30% overall identity. It alsopredicts this clone to be lacking only 11 amino acids from theN-terminus. For this reason we decided to make a chimeric constructusing the first 11 amino acids of cPLA₂ fused to the 778 amino acids ofcPLA₂-β. This construct can be used to confirm the activity of thecPLA₂-β protein.

Construction of Expression Vectors to Produce cPLA2β Protein in COS-7Cells:

An adapter was generated using synthesized oligonucleotides. Itssequence and encoded amino acids are shown below.    CTAGAGAATTCACCACCATGGACTACAAGGACGACGATGACAAGTCATTTATAGATCCTT (SEQ IDNO:7)   1 ---------+---------+---------+---------+---------+---------+60         TCTTAAGTGGTGGTACCTGATGTTCCTGCTGCTACTGTTCAGTAAATATCTAGGAA (SEQID NO:8)                      M  D  Y  K  D  D  D  D  K  S  F  I  D  PY - (SEQ ID NO:9)                       Flag-Tag                  cPLA2linker     ACCAGCACATTATAGCAGAGGTGTCCAGGACCTGCCTGCTCACGGTTCGTGTCCTGCAGG 61 ---------+---------+---------+---------+---------+---------+ 120    TGGTCGTGTAATATCGTCTCCACAGGTCCTGGACGGACGAGTGCCAAGCACAGGACGTCC      O  H  I  I  A  E  V  S  R  T  C  L  L  T  V  R  V  L  O  A-                  {circumflex over ( )}cPLA2 starts here    CCCATCGCCTACCCTCTAAGGACC 121 ---------+---------+----144    GGGTAGCGGATGGGAGATTCCTGGAT       H  R  L  P  S  K  DThis adapter was ligated with the largest BfaI-EcoRI fragment from clone52A (bps 1630-4183) into EcoRI/XbaI digested pEDΔC vector. The resultedclone, named pEDΔC-n48, was confirmed to contain the desired inserts byrestriction enzyme digestion and DNA sequencing. The sequence of theresulted clone is reported as SEQ ID NO:3.

Clone 52A was deposited with the American Type Culture Collection onJan. 23, 1997 as accession number XXXXX. pEDΔC-n48 was deposited withthe American Type Culture Collection on Jan. 22, 1997 as accessionnumber YYYYY.

(2) Transfection and Activity Assay:

Eight micrograms of plasmid pEDΔC-n48 was transfected into COS-7 cellson 10 cm cell culture plate using lipofectamine (GIBCO BRL) according tomanufacturer's protocol. PEDΔC vector DNA, pEMC-cPLA2, pEMCiPLA2 werealso transfected in parallel experiments. At 66 hours posttransfection,cells were washed twice with 10 ml of ice-cold TBS, scraped into 1 ml ofTBS. Cell pellets were collected, resuspended in lysis buffer (10 mMHEPES, pH 7.5, 1 mM EDTA, 0.1 mM DTT, 0.34 M sucrose, 1 mM PMSF and 1ug/ml leupeptin) and lysed in a Parr-bomb (700 psi, 10 min) on ice. Thelysate were centrifuged at 100,000 g for 1 hr at 4° C. The supernatant(cytosolic fraction) was transferred to another set of tubes and thepellets were resuspended in 0.5 volume of lysis buffer (particulatefraction). Twenty ul of the lysate or cytosolic fraction or 10 ul of theparticulate fraction were mixed on ice with 100 ul substrate containing20 uM 1-palmitoyl-2-[1-14C]-arachidonyl-L-3-Phosphotidylcholine, 80 mMglycine, pH 9.0, 200 uM Triton-X 100, 70% glycerol and 10 mM CaCl2. Thereaction was carried out at 37° C. for 15 min and the products analyzedas described (PNAS 87, pp 7708-7712, 1990).

EXAMPLE 2 Phopholipase Assays

1. sn-2 Hydrolysis Assays

A) Liposome: The lipid, e.g.1-palmitoyl-2-[¹⁴C]arachidonyl-sn-glycero-3-phosphocholine(PAPC), 55mCi/mmol, was dried under a stream of nitrogen and solubilized inethanol. The assay buffer contained 100 mM Tris-HCl pH 7, 4 mM EDTA, 4mM EGTA, 10% glycerol and 25 μM of labelled PAPC, where the volume ofethanol added was no more than 10% of the final assay volume. Thereaction was incubated for 30 minutes at 37° C. and quenched by theaddition of two volumes of heptane:isopropanol:0.5M sulfuric acid(105:20:1 v/v). Half of the organic was applied to a disposable silicagel column in a vacuum manifold positioned over a scintillation vial,and the free arachidonic was eluted by the addition of ethyl ether (1ml). The level of radioactivity was measured by liquid scintillation.

Variations on this assay replace EDTA and EGTA with 10 mM CaCl₂.

B) Mixed Micelle Basic: The lipid was dried down as in (A) and to thiswas added the assay buffer consisting of 80 mM glycine pH 9, 5 mM CaCl₂or 5 mM EDTA, 10% or 70% glycerol and 200 μM triton X-100. The mixturewas then sonicated for 30-60 seconds at 4° C. to form mixed micelles.

C) Mixed Micelle Neutral: As for (B) except 100 mM Tris-HCl pH 7 wasused instead of glycine as the buffer.

2. sn-1 Hydrolysis Assays

Sn-1 hydrolysis assays are performed as described above for sn-1hydrolysis, but using phospholipids labelled at the sn-1 substituent,e.g. 1-[¹⁴C]-palmitoyl-2-arachidonyl-sn-glycero-3-phophocholine.

Patent and literature references cited herein are incorporated byreference as if fully set forth.

1-20. (canceled)
 21. An antibody that binds to a phospholipase enzyme peptide comprising an amino acid sequence encoded by a polynucleotide comprising a nucleotide sequence selected from: (a) the nucleotide sequence of SEQ ID NO: 1; (b) a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 2; (c) a nucleotide sequence encoding a fragment of the amino acid sequence of SEQ ID NO: 2 having activity in a mixed micelle assay with 1-palmitoyl-2-[¹⁴C]-arachidonyl-phosphatidylcholine as a substrate; (d) a nucleotide sequence which hybridizes under stringent conditions of 4×SSC at 65 degrees C with the complement of the sequence of (a), (b) or (c) which encodes a peptide having activity in a mixed micelle assay with 1-palmitoyl-2-[¹⁴C]-arachidonyl-phosphatidylcholine as a substrate; (e) the nucleotide sequence of SEQ ID NO: 3; (f) a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 4; (g) a nucleotide sequence encoding a fragment of the amino acid sequence of SEQ ID NO: 4 having activity in a mixed micelle assay with 1-palmitoyl-2-[¹⁴C]-arachidonyl-phosphatidylcholine as a substrate; and (h) a nucleotide sequence which hybridizes under stringent conditions of 4×SSC at 65 degrees C with the complement of the sequence of (e), (f) or (g) which encodes a peptide having activity in a mixed micelle assay with 1-palmitoyl-2-[¹⁴C]-arachidonyl-phosphatidylcholine as a substrate.
 22. The antibody of claim 21, wherein the phospholipase enzyme peptide comprises an amino acid sequence encoded by a polynucleotide comprising a nucleotide sequence which hybridizes under stringent conditions of 4×SSC at 65 degrees C with the complement of a nucleotide sequence selected from: (a) the nucleotide sequence of SEQ ID NO: 1; and (b) a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:
 2. 23. The antibody of claim 21, wherein the phospholipase enzyme peptide comprises an amino acid sequence encoded by a polynucleotide comprising a nucleotide sequence which hybridizes under stringent conditions of 4×SSC at 65 degrees C with the complement of a nucleotide sequence selected from: (a) the nucleotide sequence of SEQ ID NO: 3; and (b) a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:
 4. 24. An antibody that binds to a phospholipase enzyme peptide comprising an amino acid sequence selected from: (a) the amino acid sequence of SEQ ID NO: 2; and (b) a fragment of the amino acid sequence of SEQ ID NO: 2 having activity in a mixed micelle assay with 1-palmitoyl-2-[¹⁴C]-arachidonyl-phosphatidylcholine as a substrate. (c) the amino acid sequence of SEQ ID NO: 4; and (d) a fragment of the amino acid sequence of SEQ ID NO: 4 having activity in a mixed micelle assay with 1-palmitoyl-2-[¹⁴C]-arachidonyl-phosphatidylcholine as a substrate.
 25. The antibody of claim 24, wherein the phospholipase enzyme peptide comprises the amino acid sequence of SEQ ID NO:
 2. 26. The antibody of claim 24, wherein the phospholipase enzyme peptide comprises a fragment of the amino acid sequence of SEQ ID NO: 2 having activity in a mixed micelle assay with 1-palmitoyl-2-[¹⁴C]-arachidonyl-phosphatidylcholine as a substrate.
 27. The antibody of claim 24, wherein the phospholipase enzyme peptide comprises the amino acid sequence of SEQ ID NO:
 4. 28. The antibody of claim 24, wherein the phospholipase enzyme peptide comprises a fragment of the amino acid sequence of SEQ ID NO: 4 having activity in a mixed micelle assay with 1-palmitoyl-2-[¹⁴C]-arachidonyl-phosphatidylcholine as a substrate.
 29. The antibody of claim 21, which is a monoclonal antibody.
 30. The antibody of claim 21, which is a polyclonal antibody.
 31. The antibody of claim 24, which is a monoclonal antibody.
 32. The antibody of claim 24, which is a polyclonal antibody.
 33. A composition comprising the antibody of claim 21 and at least one pharmaceutically acceptable carrier.
 34. A composition comprising the antibody of claim 24 and at least one pharmaceutically acceptable carrier. 